P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

Abstract

We investigated the properties and regulation of P2X receptors in immortalized C8-B4 cells of cerebellar microglial origin. Resting C8-B4 cells expressed virtually no functional P2X receptors, but largely increased functional expression of P2X4 receptors within 2-6 h of entering the activated state. Using real-time polymerase chain reaction, we found that P2X4 transcripts were increased during the activated state by 2.4-fold, but this increase was not reflected by a parallel increase in total P2X4 proteins. In resting C8-B4 cells, P2X4 subunits were mainly localized within intracellular compartments, including lysosomes. We found that cell surface P2X4 receptor levels increased by 3.5-fold during the activated state. This change was accompanied by a decrease in the lysosomal pool of P2X4 proteins. We next exploited our findings with C8-B4 cells to investigate the mechanism by which antidepressants reduce P2X4 responses. We found little evidence to suggest that several antidepressants were antagonists of P2X4 receptors in C8-B4 cells. However, we found that moderate concentrations of the same antidepressants reduced P2X4 responses in activated microglia by affecting lysosomal function, which indirectly reduced cell surface P2X4 levels. In summary, our data suggest that activated C8-B4 cells express P2X4 receptors when the membrane insertion of these proteins by lysosomal secretion exceeds their removal, and that antidepressants indirectly reduce P2X4 responses by interfering with lysosomal trafficking.

Involvement of P2X4 receptors in hippocampal microglial activation afterstatus epilepticus

“…However, despite numerous attempts, P2X4R‐evoked currents could not be detected in microglia 48h after induction of SE. Activity of P2X4R is tightly regulated by a balance between rapid, activity‐dependent endocytosis, and membrane insertion of the protein (Qureshi et al, ; Toulme et al, ). The large amount of ATP released during slice processing is likely to promote the rapid endocytosis of P2X4R, precluding the recording of its activity in acute brain slices.…”

CNS-derived CCL21 is both sufficient to drive homeostatic CD4+ T cell proliferation and necessary for efficient CD4+ T cell migration into the CNS parenchyma following Toxoplasma gondii infection

“…For reverse transcriptase (RT)-polymerase chain reaction (PCR), first strand cDNA was synthesized (Amersham Biosciences, NJ) and amplified by PCR using specific primers as described (Ploix et al 2001; Schmid et al 2009). Real-time PCR was performed using the iQ5 real-time PCR detection system (Bio-Rad) and primer sequences described in (Noor et al 2010; Toulme et al 2010). The relative number of CCL21 transcripts per hypoxanthine phosphoribosyl transferase (HPRT) transcripts was determined by using calibration standards for both HPRT and CCL21 as previously described (Toulme et al 2010).…”

“…Real-time PCR was performed using the iQ5 real-time PCR detection system (Bio-Rad) and primer sequences described in (Noor et al 2010; Toulme et al 2010). The relative number of CCL21 transcripts per hypoxanthine phosphoribosyl transferase (HPRT) transcripts was determined by using calibration standards for both HPRT and CCL21 as previously described (Toulme et al 2010). Briefly, PCR fragments served as standards for calibration of quantitative PCR were purified from preparative gels with an extraction kit (QiAquick; Qiagen Germany).…”

Pharmacology of P2X receptors

“…However, in a subsequent study, amitriptyline was found to have no effect at recombinant human P2X4 receptors and to act as a noncompetitive antagonist at recombinant rat and mouse P2X4 receptors 59. Interestingly, one group did find an inhibitory effect of tricyclic antidepressants and serotonin uptake inhibitors on native P2X4 receptors in mouse cerebellar microglia,60 but they concluded that this was due to inhibition of trafficking of the P2X4 receptors to the plasma membrane, rather than antagonism of the receptor per se .…”